Porous Polysaccharide Scaffold Comprising Nano-Hydroxyapatite and Use for Bone Formation

ABSTRACT

The present invention relate to three dimensional porous polysaccharide matrices able to induce mineralisation of a tissue in osseous site, as well as in non-osseous site, in the absence of stem cells or growth factors.

FIELD OF THE INVENTION

The present invention relates to a method for preparing a porouspolysaccharide scaffold comprising hydroxyapatite, preferablynano-hydroxyapatite, that supports mineralization of tissues. Thepresent invention further provides a porous polysaccharide scaffoldobtainable by said method, and its use for bone formation.

BACKGROUND OF THE INVENTION

The topic of bone-related disorders has gained considerable attentionover the past years. The use of autologous and allograft bones has beenpopularly implemented in clinics for overcoming bone related disorders,such as bone defect. However, the use of autologous bone is known toresult in secondary trauma and allograft bone induces immune repulsion.In addition, autologous and allograft bones present serious limitationssince their uses are dependent on the size and the localisation of thedefect. For example, it was reported that grafts in large defects wereresorbed by the body before the completion of osteogenesis, which leavesa doubt about the success of this therapy (Hoexter D L. Boneregeneration graft materials J Oral Implantol. 2002; 28(6); Delloye C,Cornu O, Druez V, Barbier O. Bone allografts: What they can offer andwhat they cannot. J Bone Joint Surg Br. 2007 May; 89(5):574-9).

To remedy to those drawbacks, many works have focus their interest intoreplacing natural bone by synthetically prepared implants, capable ofinducing mineralisation and of supporting new bone formation. Threedimensional scaffolds have thus been explored to repair tissues that donot self develop spontaneously. Thus, scaffold-based tissues engineeringhas become a promising strategy in regenerative medicine, because cellsalone lack the ability to form three dimensional tissues without thesupport of an artificial structure.

Prior art discloses porous scaffolds suitable for tissue engineeringsince their porous structure promotes cell colonization and tissueformation within the scaffold.

However, using said scaffolds for the treatment of bone relateddisorders still present various drawbacks related to the disease to betreated, as it depends on the type, size, and localisation of thedamaged bone, as well as on the nature, age and sex of the subject to betreated.

Currently, many works are based on the use of bioactive andbiocompatible material such as hydroxypatite. Indeed, hydroxyapatite,which is able to bond with the bone, is used as a filler to replaceamputated bone or as a coating to promote bone ingrowth into prostheticimplants. However, the use of hydroxyapatite presents limitations sinceit is mainly effective on osseous sites.

There is currently no available technique providing bone formation whichdoes not present any risk of rejection and which may be independent ofthe size and localisation of the bone to regenerate.

Consequently, there is a need for a biocompatible porous material, whichcan be used on any subject, independently of the type, size andlocalisation of the damaged bone, and is capable of promoting boneformation and providing osteoinductive properties.

SUMMARY OF THE INVENTION

The inventors have prepared porous three-dimensional polysaccharidescaffold able to provide an ideal environment for bone formation andfacilitate the growth of vasculature into the material. Surprisingly andunexpectedly, the inventors have shown that polysaccharide scaffoldcomprising nanocristalline hydroxyapatite induce mineralisation of atissue. Thus, by stimulating undifferentiated cells in situ into bonecell lineages, the invention overcomes the limitations of the prior artstrategies of treatment of bone related disorders.

The inventors have thus found out very promising polysaccharidescaffolds for bone formation, in a non-osseous site, in the absence ofgrowth factors or stem cells. The invention hence challenges thecurrently acknowledged techniques for treating bone related disordersand offers a wide range of possibilities disclosed hereafter.

The invention relates to a method for preparing a porous polysaccharidescaffold comprising the following step:

i) preparing an alkaline aqueous solution comprising an amount of atleast one polysaccharide, an amount of a cross-linking agent and anamount of a porogen agent,

ii) transforming the solution into a hydrogel by placing said solutionat a temperature from about 4° C. to about 80° C. for a sufficient timeto allow the cross-linking of said amount of polysaccharide,

iii) submerging said hydrogel into a solvent, preferably an aqueoussolution, and

iv) washing the porous polysaccharide scaffold obtained at step iii),

wherein the alkaline aqueous solution of step i) further compriseshydroxyapatite, preferably nano-hydroxyapatite.

The invention also relates to a method for preparing a porouspolysaccharide scaffold comprising the following steps:

a) preparing an alkaline aqueous solution comprising an amount of atleast one polysaccharide and one cross-linking agent,

b) freezing the aqueous solution of step a),

c) sublimating the frozen solution of step b), wherein the alkalineaqueous solution of step a) further comprises hydroxyapatite, preferablynano-hydroxyapatite,

and wherein step b) is performed before the cross-linking of thepolysaccharide occurs in the solution of step a).

The invention further relates to a porous polysaccharide scaffoldobtainable by the method of the invention.

The invention further relates to a porous polysaccharide scaffoldobtainable according to the method of the invention, for use in thetreatment of bone related disorders.

DETAILED DESCRIPTION OF THE INVENTION Definition

As used herein, the term “polysaccharide” refers to a moleculecomprising two or more monosaccharide units.

As used herein, the term “alkaline solution” refers to a solution havinga pH strictly superior to 7.

As used herein, the term “aqueous solution” refers to a solution inwhich the solvent is water.

As used herein, the term “porogen agent” refers to any solid agent whichhas the ability to form pores within a solid structure.

As used herein, the term “cross-linking” refers to the linking of onepolysaccharide chain to another one with covalent bonds.

As used herein, the term “cross-linking agent” encompasses any agentable to introduce cross-links between the chains of the polysaccharidesof the invention.

As used herein, the term “scaffold” or “matrix” refers to a semi-solidsystem comprising a three-dimensional network of one or more species ofpolysaccharide chains. Depending on the properties of the polysaccharide(or mixtures of polysaccharides) used, as well as on the nature anddensity of the network, such structures in equilibrium can comprisevarious amounts of water. In the following, the terms “scaffold” and“matrix” are interchangeable.

As used herein, the term ‘hydroxyapatite’, or “micro-hydroxyapatite” or“HA” refers to a naturally occurring mineral form of calcium apatitewith the formula Ca₅(PO₄)₃(OH), but is usually written Ca₁₀(PO₄)₆(OH)₂to denote that the crystal unit cell comprises two entities. The OH⁻ ioncan be replaced by fluoride, chloride or carbonate, producingfluorapatite or chlorapatite. Preferably, for the purpose of theinvention, the OH⁻ is not replaced. Hydoxyapatite is the major componentof bone and teeth matrix and gives bones and teeth their rigidity.Typically, the size of the microparticles of hydroxyapatite is comprisedbetween 1 to 20 μm, preferably 5 and 15 μm.

As used herein, the term “nanocristalline hydroxyapatite”, or“nano-hydroxyapatite”, or “n-HA”, refers to hydroxyapatite crystalparticles having a size comprised between 10 and 100 nm, preferably 20and 80 nm, preferably 30 and 70 nm, preferably between 30 and 60 nm, andmost preferably about 50 nm. Preferably, the n-HA particles areneedle-shaped. Preferably, the n-HA suitable for carrying out thepresent invention is a n-HA prepared by chemical precipitation at roomtemperature, for example by precipitation of a solution of phosphoricacid with a solution of calcium hydroxide.

As used herein, the term “porous composite polysaccharide scaffold”refers to a porous scaffold comprising polysaccharides associated withn-HA according to the invention.

As used herein, the term “biodegradable” refers to materials thatdegrade in vivo to non-toxic compounds, which can be excreted or furthermetabolized.

As used herein, the term “sublimation” refers to the physical phasetransition from a solid state directly to a vapor state. Morespecifically, sublimation is a process in which a substance goes from asolid to a gas without going through a liquid phase. Sublimation of asolution may be obtained through the freeze-drying process.

As used herein, the term “freeze-drying” refers the drying of adeep-frozen material under high vacuum by freezing out the solvent (ie.water) and then evaporating it in the frozen state.

As used herein, the terms “treating”, “treatment” and “therapy” refer totherapeutic treatment and prophylactic, or preventative manipulations,or manipulations which stimulate bone cell differentiation or boneformation. Such expression also encompasses manipulations which postponethe development of bone disorder symptoms, and/or reduce the severity ofbone disorders and/or such symptoms that will or are expected to developfrom a bone disorder. The terms further include ameliorating existingbone disorder symptoms, preventing additional symptoms, or preventing orpromoting bone growth.

As used herein, the expression “bone tissue” refers to calcified tissues(e.g., calvariae, tibiae, femurs, vertebrae, teeth), bone trabeculae,the bone marrow cavity, the cortical bone, which covers the outerperipheries of the bone trabeculae and the bone marrow cavity, and thelike. The expression “bone tissue” also encompasses bone cells that aregenerally located within a matrix of mineralized collagen; blood vesselsthat provide nutrition for the bone cells; bone marrow aspirates: jointfluids: bone cells that are derived from bone tissues; and may includefatty bone marrow. Finally, bone tissue includes bone products such aswhole bones, sections of whole bone, bone chips, bone powder, bonetissue biopsy, collagen preparations, or mixtures thereof. For thepurposes of the present invention, the term “bone tissue” is used toencompass all of the aforementioned bone tissues and products, whetherhuman or animal, unless stated otherwise.

As used herein, the expression “bone-related disorders” includesdisorders of bone formation and bone resorption. Preferably, theexpression “bone related disorders” refers to diseases associated withinsufficiency of bone formation or bone loss.

Non-limiting examples of bone related disorders are rickets,osteoporosis osteomalacia, osteopenia, bone cancer, arthritis, rickets,bone fracture, bone defects, osteolytic bone disease, osteomalacia, bonefrailty, loss of bone mineral density achondroplasia, cleidocranialdysostosis, Paget's disease, osteogenesis imperfecta, osteopetrosis,sclerotic lesions, pseudoarthrosis, periodontal disease, anti-epilepticdrug induced bone loss, weightlessness induced bone loss, postmenopausalbone loss, osteoarthritis, infiltrative disorders of bone, metabolicbone diseases, organ transplant related bone loss, adolescent idiopathicscoliosis, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, malnutrition, calcium deficiency, rheumatoidarthritis, hypogonadism, HIV associated bone loss, tumor-induced boneloss, cancer-related bone loss, hormone ablative bone loss, multiplemyeloma drug-induced bone loss, facial bone loss associated with aging,cranial bone loss associated with aging, jaw bone loss associated withaging, skull bone loss associated with aging, and bone loss associatedwith space travel.

Preferably, the bone related disorders, as used herein, are bonefracture, large bone defects, rickets, osteoporosis, osteogenesisimperfecta, osteomalacia, osteopenia, bone cancer, osteolytic bonedisease, bone frailty and/or loss of bone mineral density.

Porous Polysaccharide Scaffolds and Methods for Preparing Thereof

In a first object, the invention relates to a method for preparing aporous polysaccharide scaffold comprising the following step:

i) preparing an alkaline aqueous solution comprising an amount of atleast one polysaccharide, an amount of a cross-linking agent and anamount of a porogen agent,

ii) transforming the solution into a hydrogel by placing said solutionat a temperature from about 4° C. to about 80° C. for a sufficient timeto allow the cross-linking of said amount of polysaccharide,

iii) submerging said hydrogel into a solvent, preferably an aqueoussolution, and

iv) washing the porous polysaccharide scaffold obtained at step iii),wherein the alkaline aqueous solution of step i) further compriseshydroxyapatite, preferably nano-hydroxyapatite.

The concentration of the porogen agent affects both the total porosityand the size of the pores formed in the scaffolds, so that the porosityand the pore size can be under the control of the concentration of saidporogen agent.

Non-limiting examples of porogen agents are sodium chloride, calciumchloride, ammonium carbonate, ammonium bicarbonate, calcium carbonate,sodium carbonate, and sodium bicarbonate and mixtures thereof. Many ofthese compounds are available commercially from companies such asSigma-Aldrich (St. Louis, Mich., US).

Preferably, in the context of the present invention, the porogen agentis chosen from sodium chloride, calcium chloride or mixtures thereof.

Alternatively, the porogen agent may be an inorganic salt that can bedissolved once the cross-linked polysaccharide scaffold is immersed inwater. An example of such a porogen agent includes saturated saltsolution, which would be dissolved progressively.

Typically, the weight ratio of the polysaccharide to the porogen agentis in a range 1:50 to 50:1, preferably from 1:30 to 30:1, preferablyfrom 1:12 to 12:1. In a preferred embodiment, said weight ratio of thepolysaccharide to the porogen agent is about 12:14.

Typically, the aqueous solution of step iii) is water.

Alternatively, the aqueous solution of step iii) is a buffer solution.Non-limiting examples of buffer solution are PBS (Phosphate bufferedsaline), EDTA (ethylenediaminetetraacetic acid), TAPS(3-{[tris(hydroxymethyl)methyl]amino} propanesulfonic acid), Bicine(N,N-bis(2-hydroxyethyl)glycine), Tris (tris(hydroxymethyl)methylamine),Tricine (N-tris(hydroxymethyl)methylglycine), HEPES(4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid), Cacodylate (dimethylarsinicacid), SSC (saline sodium citrate), MES (2-(N-morpholino)ethanesulfonicacid) and mixtures thereof.

Alternatively, the aqueous solution of step iii) is an acidic solution.The acid may be selected from the group consisting of citric acid,hydrochloric acid, acetic acid, formic acid, tartaric acid, salicylicacid, benzoic acid, and glutamic acid.

Preferably, the aqueous solution of step iii) is a buffer solution. Mostpreferably, the aqueous solution of step iii) is phosphate buffer saline(PBS).

Preferably, the solvent of step ii) is an inorganic solvent.

In one embodiment, the method of the invention may comprise a furtherstep, consisting of freeze-drying the scaffold obtained at step iv).Freeze-drying may be performed with any apparatus known in the art.There are essentially three categories of freeze dryers: rotaryevaporators, manifold freeze dryers, and tray freeze dryers. Suchapparatus are well known in the art and are commercially available suchas a freeze-dryer Lyovac (GT2, STERIS Rotary vane pump, BOC EDWARDS).Basically, the vacuum of the chamber is from 0.1 mBar to about 6.5 mBar.The freeze-drying is performed for a sufficient time sufficient toremove at least 98.5 of the water, preferably at least 99% of the water,more preferably at least 99.5%.

In another embodiment, the method of the invention may comprise afurther step consisting of hydrating the scaffold as prepared accordingto the invention. Said hydration may be performed by submerging thescaffold in an aqueous solution (e.g., de-ionized water, water filteredvia reverse osmosis, a saline solution, or an aqueous solutioncontaining a suitable active ingredient) for an amount of timesufficient to produce a scaffold having the desired water content.Typically, when a scaffold comprising the maximum water content isdesired, the scaffold is submerged in the aqueous solution for an amountof time sufficient to allow the scaffold to swell to its maximum size orvolume. Typically, the scaffold is submerged in the aqueous solution forat least about 1 hour, preferably at least about 2 hours, and morepreferably about 4 hours to about 24 hours. It is understood that theamount of time necessary to hydrate the scaffold to the desired levelwill depend upon several factors, such as the composition of the usedpolysaccharides, the size (e.g., thickness) of the scaffold, and thetemperature of the aqueous solution, as well as other factors.

Preferably, the hydrated scaffold comprises more than 80% of water,preferably 90% of water, most preferably 95% of water.

In a second aspect, the invention relates to a method for preparing aporous polysaccharide scaffold comprising the following steps:

a) preparing an alkaline aqueous solution comprising an amount of atleast one polysaccharide, and one cross-linking agent,

b) freezing the aqueous solution of step a),

c) sublimating the frozen solution of step b), wherein the alkalineaqueous solution of step a) further comprises hydroxyapatite, preferablynano-hydroxyapatite, and wherein step b) is performed before thecross-linking of the polysaccharide occurs in the solution of step a).

It is an essential feature of the invention that step b) is performedbefore the cross-linking of the polysaccharide occurs in the solution ofstep a). Typically, temperature and time are the main factors to controlthe cross-linking of the aqueous solution. To avoid or to seriouslylimit the cross-linking of the polysaccharide, the aqueous solution maybe prepared at a temperature under 37° C., more preferably comprisedbetween 4° C. and 25° C. Moreover, the step b) may be performed asquickly as possible to avoid the cross-linking of said polysaccharide.

Once the aqueous solution is prepared, it is frozen. The freezing of theaqueous solution may be performed at different rates (e.g., ° C./min).Typically, the freezing may be performed at rate from about 1° C./min toabout 200° C./min, preferably from about 1° C./min to about 20° C./min,and most preferably from about 5° C./min to about 10° C./min. Thesolution may be frozen in liquid nitrogen or in dried ice.

When the aqueous solution is frozen, sublimation may take place. In apreferred embodiment, the method for preparing porous polysaccharidescaffolds according to the present invention includes a freeze-dryingprocess. Therefore, according to the invention, the freeze-dryingprocess has to take place before the cross-linking process occurs in theaqueous solution. Freeze-drying may be performed with any apparatusknown in the art. There are essentially three categories of freezedryers: rotary evaporators, manifold freeze dryers, and tray freezedryers. Such apparatus are well known in the art and are commerciallyavailable such as a freeze-dryer Lyovac (GT2, STERIS Rotary vane pump,BOC EDWARDS). Basically, the deep-frozen aqueous solution is placed in achamber. Then the chamber temperature is increased to a level higherthan the boiling point of the liquefied vapour, whereby the vapour isvaporized and removed. Typically, the temperature of chamber may be from−70° C. to −1° C., preferably from −70° C. to −40° C., furtherpreferably about −50° C. to −40° C. The heating of the chamber isaccompanied with a vacuum flow to decrease the pressure of the chamber.Typically, the vacuum of the chamber is from 0.1 mBar to about 6.5 mBar.Typically, the freeze-drying is performed for a sufficient timesufficient to remove at least 98.5% of the water, preferably at least99% of the water, more preferably at least 99.5%.

The freezing of the aqueous solution causes the formation of iceparticles from the water. Without to be bound by any theory, under thetemperature and pressure condition described above, water included inthe frozen solution is sublimed, and thus, thereby leaving intersticesin the material in the spaces previously occupied by the ice particles,and accordingly porous polysaccharide scaffolds are produced.Surprisingly, the cross-linking process occurs during the freeze-dryingprocess.

The material density and pore size of the resultant scaffold may betherefore varied by controlling the rate of freeze-drying of the frozenaqueous solution. The essential parameter in a freeze-drying process isthe vacuum rate.

For the purpose of the present invention, any type of polysaccharide canbe used. Synthetic or natural polysaccharide may be alternatively usedin the context of the invention. Non-limiting examples of suitablepolysaccharide for implementing the present invention are dextran, agar,alginic acid, hyaluronic acid, inulin, pullulan, heparin, fucoidan,chitosan, scleroglucan, curdlan, starch, cellulose and mixtures thereof.Chemically modified polysaccharides bearing for instance acidic groups(carboxylate, sulphate, phosphate), amino groups (ethylene amine,diethylaminoethylamine, propylamine), hydrophobic groups (alkyl, benzyl)can be included. Saccharide structures and oligosaccharides that may beused to produce the desired materials include but are not limited toribose, glucose, mannose, galactose, fructose, sorbose, sorbitol,mannitol, iditol, dulcitol and mixtures thereof. Many of these compoundsare available commercially from companies such as Sigma-Aldrich (St.Louis, Mich., US).

Typically, the average molecular weight of the polysaccharides is fromabout 5,000 Daltons to about 2,000,000 Daltons, preferably from about100,000 Daltons to about 500,000 Daltons. Typically, the polysaccharideused to prepare the scaffold of the invention is a neutralpolysaccharide such as dextran, agar, pullulan, inulin, scleroglucan,curdlan, starch, cellulose and mixtures thereof. Alternatively, thepolysaccharide used to prepare the scaffold of the invention is apositively charged polysaccharide such as chitosan, DEAE-dextran,DEAE-pullulan, EA-pullulan and mixtures thereof. Alternatively, thepolysaccharide used to prepare the scaffold of the invention is anegatively charged polysaccharide such as alginic acid, hyaluronic acid,heparin, fucoidan and mixtures thereof. Alternatively, thepolysaccharide used to prepare the scaffold of the invention is amixture of neutral and negatively charged polysaccharides. Typically,the negatively charged polysaccharides represent 1 to 20%, preferably 5to 10% of the mixture. Alternatively, the polysaccharide used to preparethe scaffold of the invention is a mixture of neutral and positivelycharged polysaccharides. Typically, the positively chargedpolysaccharides represent 1 to 20%, preferably 5 to 10% of the mixture.

Preferably, for the purpose of the invention, said polysaccharide isselected in the group consisting of dextran, pullulan, agar, alginicacid, starch, hyaluronic acid, inulin, heparin, fucoidan, chitosan andmixtures thereof. In one particular embodiment of the invention, saidpolysaccharide is a mixture of pullulan and dextran. Typically, theweight ratio of pullulan/dextran is in a range from 95:5 to 95:5 (w/w),preferably in a ration of 75:25 (w/w). In another embodiment of theinvention, said polysaccharide is a mixture of pullulan, dextran andfucoidan. Typically, the weight ratio of pullulan/dextran/fucoidan is ina range from about 70:20:10 to about 50:20:30, preferably from about70:20:10 to about 50:30:20, and most preferably in a ratio of about73:22:5 (w/w). The presence of fucoidan in the porous polysaccharidescaffold of the invention is highly advantageous since fucoidan promotesvascularisation.

Typically, the covalent cross-linking agent is selected from the groupconsisting of trisodium trimetaphosphate (STMP), phosphorus oxychloride(POCl₃), epichlorohydrin, formaldehydes, carbodiimides, glutaraldehydes,any other compound that is suitable for crosslinking a polysaccharideand mixtures thereof. Many of these compounds are available commerciallyfrom companies such as Sigma-Aldrich (St. Louis, Mich., US). Preferably,for the purpose of the present invention, said cross-linking agent isSTMP. Typically, the concentration of the covalent cross-linking agentin the aqueous solution (w/v) is from about 1% to about 6%, morepreferably from about 2% to about 6%, most preferably from about 2% toabout 3%. Typically, the weight ratio of the polysaccharide to thecross-linking agent is in a range from 20:1 to 1:1, preferably from 10:1to 2:1.

In the context of the present invention, nano-hydroxyapatite may be acommercial nano-hydroxyapatite, such as those commercialised by InframatCorporation or Fluidinova. Preferably, nanocristalline hydroxyapatiteuseful in the context of the present invention is obtained throughchemical precipitation at room temperature of a solution of phosphoricacid, at a concentration comprised between 0.3 to 1M, preferably 0.6M,with a solution of calcium hydroxide, at a concentration comprisedbetween 0.5 to 1.5M, preferably 1M. Typically, the concentration ofhydroxyapatite in the alkaline solution of polysaccharide (w/v) iscomprised between 0.01 and 10% (w/v), preferably between 0.1 and 0.5%(w/v), more preferably between 0.1 and 0.3% (w/v). Typically, theconcentration of nano-hydroxyapatite in the alkaline solution ofpolysaccharide (w/v) is comprised between 0.01 and 10% (w/v), preferablybetween 0.1 and 0.5% (w/v), more preferably between 0.1 and 0.3% (w/v).

In one embodiment, the alkaline aqueous solution of step a) or step i)comprising hydroxyapatite, preferably nano-hydroxyapatite, may be pouredin a mould before step b) or step ii), so that the porous polysaccharidescaffold obtained with the method of the invention can take a desiredform. Any geometrical moulds may be used according to the invention.Different sizes may also be envisaged. The mould may be made of anymaterial, but preferred material includes non sticky surfaces such asTeflon.

Alternatively, the scaffolds of the invention may be cut and shaped totake a desired size and form.

The methods of the invention can further include the step of sterilizingthe scaffold using any suitable process. The scaffold can be sterilizedat any suitable point, but preferably is sterilized before the scaffoldis hydrated. A suitable irradiative sterilization technique is forexample an irradiation with Cesium 137, 35 Gray for 10 minutes. Suitablenon-irradiative sterilization techniques include, but are not limitedto, UV-exposure, gas plasma or ethylene oxide methods known in the art.For example, the scaffold can be sterilized using a sterilisation systemwhich is available from Abtox, Inc of Mundelein, Ill. under the trademark PlazLyte, or in accordance with the gas plasma sterilizationprocesses disclosed in U.S. Pat. No. 5,413,760 and U.S. Pat. No.5,603,895.

The scaffold produced by the methods of the invention can be packaged inany suitable packaging material. Desirably, the packaging materialmaintains the sterility of the scaffold until the packaging material isbreached.

In a further embodiment, the alkaline solution of step i) or a) furthercomprises a drug. The invention thus provides porous polysaccharidescaffold comprising a drug. Typically, said drug is a drug having anacknowledged therapeutic effect, such as hormones radioactive substance,fluorescent substance, chemotactic agent, antibiotic, steroidal ornon-steroidal analgesic, immunosuppressant, or anti-cancer drug, drugsbelonging to the pharmaceutical class of statins. Preferably, said drugbelongs to the pharmaceutical class of statins. As used herein,“statins” refers to a pharmaceutical class of HMG-CoA reductaseinhibitors. It has been recently shown that some of the drugs from thispharmaceutical class play a role in the process of bone formation.Preferably, said statins is selected from the group consisting oflovastatin, atorvastatin, mevastatin pitavastatin, rosuvastatin,pravastatin, fluvastatin and simvastatin. More preferably, said statinsis selected from the group consisting of lovastatin, atorvastatin,mevastatin and simvastatin. Said statins are highly appropriate in thecontext of the present invention since they play a role in the boneformation.

In a further embodiment, the alkaline solution further comprises abioactive substance. Typically, said bioactive substance is a substanceknown for playing an important role in various mechanisms such asmodification of cellular pathways and modification of cellular ortissular responses. Said bioactive substance is chosen among growthfactors, cytokines (lymphokines, interleukins, and chemokines),antioxidant molecules, angiogenic molecule, anti-angiogenic agents,immunomodulating agents, proinflammatory cytokines, anti inflammatorycytokines, plasma-derived bioactive substances, PRP (platelet richplasma)-derived substances, soluble adhesion molecules.

In a third aspect, the invention relates to porous polysaccharidescaffolds obtainable by the methods of the invention. These porouspolysaccharide scaffolds are indeed the only ones which have theremarkable properties provided by the invention. When the method ofpreparing the porous polysaccharide scaffold according to the inventioninvolves the use of a porogen agent, the concentration of the porogenagent affects the size of the pores formed in the scaffolds. Therefore,in this particular embodiment, the size of the pores can be under thecontrol of the concentration of said porogen agent. Typically, theaverage pore size of the scaffold is from about 1 μm to about 500 μm,preferably from about 10 μm to about 200 μm. Typically, the density ofthe pores (or porosity) is from about 4% to about 75%, preferably fromabout 4% to about 50%. The person skilled in the art may provide desiredproperties to the porous polysaccharide scaffold according to theinvention. Typically, the person skilled in the art may add one or morecompounds chosen in the group consisting of a biomolecule, a bioactiveagent, a drug, an anti-inflammatory agent, an additive, an antimicrobialagent, a colorant, a surfactant and a differentiation agent. Thetechniques for incorporating said compounds in the porous polysaccharidescaffold of the invention completely falls within the ability of theperson skilled in the art. Typically, said compounds may be addeddirectly the alkaline solution of step i) or a) of the method of theinvention. In this particular embodiment, the compound would be withinthe structure of the porous polysaccharide scaffold of the invention.Alternatively, said compounds can be incorporated into the porouspolysaccharide scaffold during a step consisting of hydrating saidscaffold with a solution of the compound.

In one embodiment, the porous polysaccharide scaffold of the inventionfurther comprises one or more biomolecules. Non-limiting examples ofbiomolecules are drugs, hormones, radioactive substances, fluorescentsubstances, chemicals or agents, chemotactic agents, antibiotics,steroidal or non-steroidal analgesics, immunosuppressants, anti-cancerdrugs, short chain peptides, glycoprotein, lipoprotein, cell attachmentmediators, biologically active ligands, integrin binding sequence,ligands, small molecules that affect the up-regulation of specificgrowth factors, tenascin-C, hyaluronic acid, chondroitin sulphate,fibronectin, decorin, thromboelastin, thrombin-derived peptides, andmixtures thereof. The presence of said biomolecules in the porouspolysaccharide scaffold of the invention may enhance treatment effects,enhance visualization, indicate proper orientation, resist infection,promote healing, may increase softness or any other desirable effects.In another embodiment, the porous polysaccharide scaffold of theinvention further comprises a bioactive substance. Typically, saidbioactive substance is a substance known for playing an important rolein various mechanisms such as modification of cellular pathways andmodification of cellular or tissular responses. Said bioactive substanceis chosen among growth factors, cytokines (lymphokines, interleukins,and chemokines), antioxidant molecules, angiogenic molecule,anti-angiogenic agents, immunomodulating agents, proinflammatorycytokines, antiinflammatory cytokines, plasma-derived bioactivesubstances, PRP (platelet rich plasma)-derived substances, and solubleadhesion molecules.

In a further embodiment, the porous polysaccharide scaffold of theinvention further comprises one or more drug. Typically, said drug is adrug having an acknowledged therapeutic effect, such as hormonesradioactive substance, fluorescent substance, chemotactic agent,antibiotic, steroidal or non-steroidal analgesic, immunosuppressant, oranti-cancer drug, drugs belonging to the pharmaceutical class ofstatins. Preferably, said drug belongs to the pharmaceutical class ofstatins. Preferably, said statins is selected from the group consistingof lovastatin, atorvastatin, mevastatin pitavastatin, rosuvastatin,pravastatin, fluvastatin and simvastatin. More preferably, said statinsis selected from the group consisting of lovastatin, atorvastatin,mevastatin and simvastatin. Said statins are highly appropriate in thecontext of the present invention since they play a role in the boneformation

In another embodiment, the porous polysaccharide scaffold of theinvention further comprises anti-inflammatory agents. Non-limitingexamples of anti-inflammatory agents are indomethacin, salicylic acidacetate, ibuprofen, sulindac, piroxicam, and naproxen; thrombogenicagents, such as thrombin, fibrinogen, homocysteine, and estramustine;and radio-opaque compounds, such as barium sulfate, gold particles andiron oxide nanoparticles (USPIOs) and mixtures thereof.

In still another embodiment, the porous polysaccharide scaffold of theinvention further comprises additives. The amount of the additive useddepends on the particular application of the porous polysaccharidescaffold of the invention and may be readily determined by the personskilled in the art using routine experimentation.

In still another embodiment, the porous polysaccharide scaffold of theinvention further comprises an antimicrobial agent. Suitableantimicrobial agents are well known in the art. Non-limiting examples ofsuitable antimicrobial agents are alkyl parabens, such as methylparaben,ethylparaben, propylparaben, and butylparaben; cresol; chlorocresol;hydroquinone; sodium benzoate; potassium benzoate; triclosan andchlorhexidine and mixture thereof. Other examples of antibacterialagents and of anti-infectious agents that may be used are, in anon-limiting manner, rifampicin, minocycline, chlorhexidine, silver ionagents and silver-based compositions and mixtures thereof.

In a further embodiment, the porous polysaccharide scaffold of theinvention further comprises at least one colorant to enhance thevisibility of the scaffold. Suitable colorants include dyes, pigments,and natural coloring agents. Non-limiting examples of suitable colorantsare alcian blue, fluorescein isothiocyanate (FITC) and FITC dextran andmixtures thereof.

In still another embodiment, the porous polysaccharide scaffold of theinvention further comprises at least one surfactant. Surfactant, as usedherein, refers to a compound that lowers the surface tension of water.The surfactant may be an ionic surfactant, such as sodium laurylsulfate, or a neutral surfactant, such as polyoxyethylene ethers,polyoxyethylene esters, and polyoxyethylene sorbitan and mixturesthereof.

In one embodiment, the porous polysaccharide scaffold of the inventionfurther comprises a differentiation agent. Preferably, such adifferentiation agent is an agent involved in bone formation.Alternatively, such a differentiation agent is an agent involved inosteogenesis, angiogenesis or wound healing. Preferably, such adifferentiation agent is a growth factor. Non-limiting examples ofgrowth factor suitable for the purpose of the present invention areepidermal growth factor (EGF), insulin-like growth factor (IGF-I,IGF-II), transforming growth factor beta (TGFβ), heparin binding growthfactor (HBGF), stromal derived factor (SDF-1), vascular endothelialgrowth factors (VEGF), fibroblast growth factors (FGFs), plateletderived growth factors (PDGF), parathyroid hormone (PTH), parathyroidhormone related peptide (PTHrP), basic fibroblast growth factor (bFGF);TGFβ superfamily factors; bone morphogenetic proteins (BMPs) preferablyBMP2, BMP3, BMP4, BMP5, BMP7, somatropin, growth differentiation factor(GDF) and mixtures thereof.

Typically, the growth factor is present at a concentration comprisedfrom 1 ng to 100 μg per porous polysaccharide scaffold of the invention.

In another embodiment, the porous polysaccharide scaffold of theinvention further comprises cells, such as yeast cells, mammalian cells,insect cells, and plant cells.

Preferably, said cell is a mammalian cell. Non-limiting examples ofmammalian cells suitable for the purpose of the invention aredifferentiated cells such as chondrocytes, fibrochondrocytes,osteocytes, osteoblasts, osteoclasts, synoviocytes, epithelial cells andhepatocytes or stem cells, embryonic stem cells, induced progenitor stemcells (iPS), mesenchymal stem cells from different sources, bone marrow,adipose tissue, peripheral blood progenitor cells, cord blood progenitorcells, genetically transformed cells and mixtures thereof. Mostpreferably, the mammalian cells comprised in the porous polysaccharidescaffold according to the invention are adipose derived stroma cells.Typically, the mammalian cells comprised in the porous polysaccharidescaffold are present at a cell density comprised between 200 cells/mm³to 35 000 cells/mm³.

In a fourth aspect, the invention relates to a porous polysaccharidescaffold obtainable according to the method of the invention for use forbone generation.

As used herein, the expression “bone generation” encompasses “bonerepair” and “bone development”.

In a fifth aspect, the invention relates to a porous polysaccharidescaffold obtainable according to the method of the invention for use forstimulating ectopic mineralized tissue formation. In the context of thepresent invention, the expression “ectopic” refers to a non osseoustissue. Therefore, the invention also relates to a porous polysaccharidescaffold obtainable according to the method of the invention for use forinducing mineralized tissue in a non-osseous site.

Preferably, said stimulation of ectopic mineralization occurs in absenceof stem cells and/or growth factors. Indeed, the inventors have shownthat the porous polysaccharide scaffold according to the invention hasthe ability to induce mineralized tissue in a non-osseous site and in anosseous site (calvaria site or femoral condyle), even in the absence ofstem cells and/or growth factors. Therefore, the invention provides aporous polysaccharide scaffold useful for stimulating mineralized tissueformation in osseous site, as well as in non-osseous site, in thepresence as well as in the absence of stem cells and/or growth factors.

Use of the Porous Polysaccharide Scaffold According to the Invention

The inventors have shown that implanting porous polysaccharide scaffoldaccording to the invention lead to the stimulation of a dense collagennetwork and blood vessel formation as well as the recruitment ofosteoblast-like cells. Said implantation of scaffolds according to theinvention in subcutaneous site leads to the formation of a densemineralized tissue, and thus to bone formation.

The inventors have shown that the scaffold of the invention, whenimplanted, retains growth factor such as VEGF and BMP. The inventorsalso evidenced that the ability of retaining said growth factor washigher for the scaffold comprising n-HA, compared to a scaffold notcomprising n-HA.

In a sixth aspect, the invention relates to a porous polysaccharidescaffold obtainable according to the method of the invention for use inthe treatment of bone related disorders. The inventors have indeed shownthe ability of the porous polysaccharide scaffold according to theinvention to stimulate the production of an extracellular mineralizedmatrix, probably through differentiation of cells into bone cells. Thus,the inventors evidenced that the scaffold of the invention is useful forthe treatment of bone related disorders.

In a seventh aspect, the invention relates to a porous polysaccharidescaffold obtainable according to the method of the invention for use asa polysaccharide scaffold.

Typically, the size and the shape of the porous polysaccharide scaffoldcan be adapted to the type and size of the bone to replace, and to thelocalization of said bone. Preferably, the shape of the scaffold is asphere, a cylinder, a cube or a rectangular cuboid. Preferably, the sizeof said scaffold is comprised between 0.5 mm and 30 cm. Typically, thepolysaccharide scaffold of the invention may be is implanted as follows:the lyophilized scaffold is placed within the defect and its size isadapted to the size of defect. For example, for the implantation incalvaria site in mouse, defects of 4 mm of diameter and 500 vim of depthwere performed and the matrices were apposed onto the host tissue. Inmice, bone defect performed in the femoral condyle is around 1 mm³. Inrat, the critical size defect performed in the femoral condyle is 5 mmof diameter and 3 mm of depth. These bone defects are filled with thematrices. For segmental bone defect in large animal (sheep or goat), aresection of 2.5 cm is performed at metatarsus and cylinder ofpolysaccharide scaffold is placed within the defect. Analysis of thenewly formed tissue within the defect is performed between 15 days to 12months. The person skilled in the art is award of the routine suitabletechniques for analyzing said newly formed tissue. Typically, saidanalysis may be performed using several invasive methods such ashistomorphometry as gold standard technique. Alternatively, saidanalysis may be performed using non invasive imaging approaches such asMagnetic Resonance Imaging (MRI), X Ray micro Computed Tomography(micro-CT), Single Photon Emission Computarized Tomography (SPECT) orradiological analysis. The choice of the suitable technique is dependenton the type of bone in small and large animals, or humans.

FIGURES LEGENDS

FIG. 1: Porous polysaccharide scaffold.

Macroscopic view of hybrid porous discs with n-HA before (FIG. 1A) andafter (FIG. 1B) rehydration with phosphate buffer saline (PBS). Thescale bar corresponds to 1 mm.

FIG. 2: Electron Microscopy of a freeze-dried polysaccharide scaffold.

The morphology of freeze-dried scaffolds was analyzed by scanningelectron microscopy (FIG. 2A). After rehydration in PBS, porosity ofhydrated scaffolds was observed with Environmental Scanning ElectronMicroscopy (ESEM Philips XL 30) (FIG. 2B).

FIG. 3: Healing of critical size defects in nude mice by thepolysaccharide-based matrices.

Micro-CT images of calvaria defects filled with polysaccharide matriceswithout n-HA (FIG. 3A), or with the polysaccharide scaffold (FIG. 3B),loaded (on left side) or not (on right side) with 5×10⁵ differentiatedadipose derived stromal cells (ADSCs). Imaging on the same animal foreach type of scaffold was performed after 15, 30, 60 and 84 days ofimplantation, and resulting images are respectively referred to as D15,D30, D60, D84. Quantitative analysis of the Tissue Mineral Density (TMD)of implanted polysaccharide scaffold. Calvaria bone was used as acontrol (FIG. 3C).

FIG. 4: Ectopic mineralized tissue formation in subcutaneous siteinduced by the polysaccharide scaffold.

(A) Micro-CT images at Days 15, 30 and 60 of a mouse implanted with twodiscs of the polysaccharide scaffold (n-HA/scaffold) (left site) and onedisc previously seeded with 5×10⁵ differentiated ADSCs (right site).

(B) Macroscopic view at D60.

(C) Quantitative analysis of the tissue mineral density (TMD).

(D) Histological examination of undecalcified (D1; magnification ×10)(stained by Goldner's trichrome) and decalcified (D2; magnification ×2)(D3; magnification ×20) sections (Masson's staining) obtained at Day 60.

(E) Von Kossa staining performed on explanted materials at Day 30 andDay 60. Control was performed using the paraffin-embedded compositematrix before implantation (magnification ×2).

FIG. 5: Matrix+n-HA (MATRI+) induces mineralization in ectopic site ofmice.

(A) Representative micro-CT images of the subcutaneous implantation ofthe Matrix alone on the left side (indicated by an arrowed doted line)and Matrix+n-HA (MATRI+) on right side (indicated by an arrowed plainline), after 15 (D15), 30 (D30) and 60 days (D60) of implantation inBalb/c mice.

(B) Bone Mineral Content (BMC) and Bone Mineral Density (BMD) weremeasured from reconstructed three-dimensional micro-CT images withMicroview Image analyser of the Matrix (white rectangle) and Matrix+n-HA(MATRI+) (black rectangle). Data are presented as means±standarddeviation for n=8. The symbol ** indicates a statistically significantdifference compared to the other groups <0.01.

FIG. 6: Matrix+n-HA induces formation of a collagen-based mineralizedtissue: histological analysis of the newly formed tissue.

(A) Representative histological undecalcified sections of the Matrix andMatrix+n-HA (MATRI+) samples implanted subcutaneously in mice, after 15days (D15) and 60 days (D60): Von Kossa staining.

(B) Representative histological decalcified sections of Matrix+n-HA(MATRI+) 60 days after implantation:Goldner staining, The images showeda high dense collagen tissue around the implant that colonizes thescaffold, with osteoblast-like cells as indicated by the white arrows,and numerous vessels inside the collagen tissue indicated by the blackarrows.

FIG. 7: XRD patterns of matrices before surgery (D0) and 15 days (D15)after subcutaneously implantation in mice.

(A) Matrix+n-HA (MATRI+); (B) Matrix without n-HA

Specific peaks of hydroxyapatite (HA) are only observed in the XRDpatterns after 15 days of implantation of MATRI+. Peaks of Halite (H)due to sample processing, are observed in all spectra. The XRD patternsobtained at day 30 and day 60 are similar than those observed at D15 forboth groups (data not shown).

FIG. 8: Matrix+n-HA (MATRI+) retained endogeneous osteoinductive andangiogenic factors.

Measurement by ELISA of BMP2 (A) and VEGF₁₆₅ (B), retained in the tissueformed within the Matrix (white rectangle) and Matrix+n-HA (MATRI+)(black rectangle) when implanted subcutaneously at D15, D30 and D60.Results are expressed in pg of growth factors retained per μg ofproteins quantified by BCA. Data are presented as means±standarddeviation for n=6 samples. The symbols * and ** indicate a statisticallysignificant difference compared to the other groups with p<0.05 and<0.01, respectively.

FIG. 9: Matrix+n-HA (MATRI+) induces a high mineralization of tissue ina critical size bone defect performed in the femoral condyle of rats.

(A) Representative micro-CT images of the femoral condyle of rats, 15days (D15), 30 days (D30) and 90 days (D90) after implantation withoutscaffold (empty), with Matrix or Matrix+n-HA (MATRI+).

(B) Bone Mineral Content (BMC) and Bone Mineral Density (BMD) weremeasured from reconstructed three-dimensional micro-CT images of theempty group (white rectangle), the Matrix group (grey rectangle) andMatrix+n-HA (MATRI+) (black rectangle). Data are presented asmeans±standard deviation for n=4. The symbol ** indicates astatistically significant difference compared to the other groups withp<0.01.

FIG. 10: Matrix+nHA (MATRI+) induces a high mineralized bone tissue in acritical size bone defect performed in the femoral condyle of rats after90 days of implantation; histological analysis of the newly formedtissue.

(A) Representative histological undecalcified sections of Empty, Matrixand Matrix+n-HA (MATRI+) samples implanted in the femoral condyle ofrats, after 90 days of implantation: Von Kossa staining. The arrowsindicated the position of the bone defect.

(B) Representative histological decalcified sections of Empty, Matrixand Matrix+nHA samples 90 days after implantation:Goldner staining, Afibrous tissue was formed in the empty bone defect, while bone formationoccurred in direct contact of the matrix and was enhanced within theMATRIX+ implant.

EXAMPLE Example 1: Implantation of the Scaffold of the Invention inCalvaria Site of Athymic Mice Materials and Methods Nano-hydroxyapatitePreparation

Nano-hydroxyapatite (n-HA) was prepared by wet chemical precipitationusing a 0.6M solution of Phosphoric acid (H₃PO₄ Rectapur, Prolabo®,France) and a 1M solution of calcium hydroxide (CaOH₂ Alfa Aesar,Germany). 100 ml of H₃PO₄ solution were added dropwise in 100 ml ofCaOH₂ solution during 30 minutes under vigorous stirring at roomtemperature. At the end of reaction, pH was adjusted to 9 using 0.4.10⁻³mol of a 0.6 M sodium hydroxide solution, then stirring was continuedduring 12 hours.

Nano-hydroxyapatite (n-HA) has been characterized by transmissionelectron microscopy (TEM), scanning electron microscopy and by FTIRanalysis. TEM revealed n-HA needle-shaped crystals of 50 nm long. FTIRanalysis showed specific bands of phosphate ions of at 559 cm⁻¹, 601cm⁻¹ and 1018 cm⁻¹ and a non-specific carbonate band 1415 cm⁻¹.

Preparation of Composite Polysaccharide Scaffolds (MATRI+)

Macroporous composite scaffolds (MATRI+) were prepared using a blend ofpullulan/dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc, DextranMW 500,000, Pharmacia), prepared by dissolving 9 g of pullulan and 3 gof dextran into 27 mL of distilled water containing 14 g of NaCl and 13mL of nano-hydroxyapatite suspension (n-HA, 6.36% w/v). Chemicalcross-linking was carried out using trisodium trimetaphosphate STMP(Sigma) under alkaline condition. Briefly, 1 mL of 10M sodium hydroxidewas added to 10 g of the polysaccharide blend, followed by the additionof 1 mL of water containing 300 mg of STMP. After incubation at 50° C.for 15 min, resulting scaffolds were cut into 6 mm diameter discs,neutralized in PBS 10× (pH 7.4) then washed extensively with a 0.025%NaCl solution. After a freeze-drying step, porous compositepolysaccharide scaffolds were stored at room temperature until use.Fluorescent scaffolds were prepared by adding 1% of FluoresceinIsoThioCyanate (FITC) dextran (Sigma, St. Louis Mo., USA) to the mixturebefore cross-linking.

ADSC Cultures and Osteogenic Differentiation

Adipose Derived Stromal Cells (ADSCs) were isolated from human adiposetissue after a digestion with 0.1% (w/v) collagenase type I and culturedas previously described by Gimble et al, 2007. The remaining StromalVascular Fraction (SVF) was cultured in a basal medium (DMEM F12 medium(Invitrogen) supplemented with 10% (v/v) Foetal Bovine Serum (FBS) or inan osteogenic medium for inducing osteoblastic differentiation of ADSCs(IMDM medium (Invitrogen), supplemented with 10% (v/v) FBS (Lonza), 10⁻⁸M dexamethasone (Sigma), 50 mg/ml ascorbic acid (Sigma) and 10 mMβ-glycerophosphate (Sigma)).

Experimental Models in Nude Mice

Orthotopic new bone formation was assessed on calvaria site of athymicmice. Twelve weeks-old nude mice were anesthetized with anisoflurane/N2O mixture and were subjected to surgery to make a 4 mmdiameter full thickness on the left and right parietal bone using atrephine dental burr. Disk-shaped matrices without n-HA (Group 1) andcomposite polysaccharide scaffold MATRI+ containing n-HA (Group 2) wereimplanted on top of the periosteum of the parietal bone. Group 3corresponds to mice implanted with the composite polysaccharide scaffoldassociated with differentiated ADSCs one week before implantation.

To study ectopic bone formation, polysaccharide-based matrices (Group1), composite polysaccharidescaffold without cells (Group 2), ormatrices previously seeded with differentiated ADSCs (Group 3), wereimplanted into dorsal, subcutaneous spaces of athymic mice (female, 12weeks old). Four scaffolds were implanted by mice. Bone formation wasfollowed by a non invasive high resolution X-ray tomography (micro-CT)analysis performed 15, 30 and 60 days after implantation and byhistological examination at the end of the experiment (D60).

High Resolution X-Ray Tomography (Micro-CT) Analysis

Mice were scanned in an in vivo Explore Locus SP X-Raymicro-computerized tomography (micro-CT) device (General Electric) at anisotropic resolution of 45 Reconstruction of the parietal andsubcutaneous region was performed following correction of rotationcentre and calibration of mineral density. Bone analysis was performedusing the “Advanced Bone Analysis”™ software (GE). Thresholding of greyvalues was performed using the histogram tool in order to separatemineralized elements from background. The density of mineralized tissue(TMD) was determined in the region of interest (ROI).

Histological Evaluation

At the end of the experimental periods, mice were euthanized and sampleswere dissected out and fixed in 3.7% (v/v) paraformaldehyde in PBS 0.1MpH 7.4. One part of the samples were decalcified and embedded inparaffin. Permanent sections of 7 micron were stained with hematoxylinand eosin and Masson trichrome dye. The other part of the samples wereembedded in methylmethacrylate as described by Schenk et al, 1984.Longitudinal sections (15 μm thick) were prepared using a Leicamicrotome and tungsten carbide blades. Sections were stained withGoldner's trichrome, Von Kossa, and observed using a Nikon Eclipse 80imicroscope. Pictures were generated using a DXM 1200 C (Nikon) CCDcamera.

Results

3D porous matrices (FIG. 1) were obtained according to the methodsdisclosed in the PCT patent applications WO2009/047346 andWO2009/047347, with n-HA included in the starting formulation. n-HA insuspension (6.36% (w/v)) allowed an homogeneous dispersion of the HAnanoparticles in the resulting 3D matrices. The n-HA matrices containedin the dry state, 2.8+/−0.1% (w/w) of HA. The use of n-HA in the dryform instead of a n-HA suspension, induced large aggregates inside thematrices. The 3D matrices in the presence of n-HA are porous (FIG. 2)with pore sizes controlled by the patented process.

Discs of 4 mm in diameter of 3D porous matrices with or without n-HA(composite scaffold) and previously seeded or not with human adiposederived mesenchymal stem cells (ADSCs) were then evaluated in two micemodels.

Orthotopic new bone formation on calvariae site of athymic mice revealedthat only the polysaccharide-based matrices associated with n-HA(composite scaffold) induced formation of a mineralized tissue in nudemice. The porous matrices without n-HA do not induce any mineralizationwithin 60 days. The orthotopic new bone formation was observed withcomposite matrices in absence of human mesenchymal stem cells, and evenif the scaffold moved out of the bone defect (FIG. 3B). Themineralization occurred four weeks after implantation and increased withtime (FIG. 3C). Histological examination (Goldner's trichrome staining)revealed a fibrous tissue formed when polysaccharide-based matriceswithout n-HA were implanted, whereas the composite polysaccharidescaffold provides an efficient scaffold for local production of collagennetwork within the matrices.

Since the n-HA matrix (composite scaffold) was found to inducemineralization outside the bone defect, the inventors next examined itspotency to stimulate ectopic bone formation. They observed thatimplantation of matrices without n-HA did not form any mineralizedtissue at day 60. In contrast, implantation of n-HA matrices (compositepolysaccharide scaffold of the invention) in subcutaneous site lead tothe formation of a dense mineralized tissue (FIGS. 4A and 4B) four weeksafter implantation and without ADSCs seeding. The mineralizationincreased with time. Quantification indicated that the TMD of thecalcified tissue was about 420 mg/cm³ and close to the density of theimplanted composite matrix in orthotopic site (FIG. 4C) 60 days afterimplantation. Histological analysis on undecalcified (FIG. 4D ₁) anddecalcified (FIG. 4D ₂) sections of the ectopically induced mineralizedtissue revealed that n-HA matrices (composite polysaccharide scaffoldMATRI+) stimulated a dense collagen network and blood vessel formationas well as the recruitment of osteoblast-like cells (FIG. 4D ₃). Tovisualize the level of calcification in the newly formed tissue,sections of n-HA/scaffold were stained according to Von Kossa techniqueat day 30 and day 60 (FIG. 4E). Controls were performed on theparaffin-embedded composite polysaccharide. This staining showed awell-calcified tissue of n-HA/scaffold that increases with time ofimplantation. To the knowledge of the inventors, no material so far inthe absence of stem cells or growth factors, was able to give thiseffect.

The inventors further investigated for comparison the role of n-HA aloneon non-osseous site. For this purpose, they proceed to the implantationof n-HA alone in subcutaneous site. After 15 days and 30 days, they onlyobserved a classical reaction to a foreign body. Indeed, thehistological examination of undecalcified section (Cyanine Solochromestaining) of non-osseous site implanted with n-HA alone did not show thepresence of any mineralized tissue. Implantation of n-HA alone hence didnot lead to the formation of a mineralized tissue.

The inventors have thus shown that the porous composite polysaccharidescaffold of the invention provides unexpected results by stimulatingmineralized tissue formation in osseous site, as well as in non-osseoussite, in the absence of stem cells or growth factors.

Example 2: Implantation of the Scaffold According to the Invention in aNon Osseous Site in Mice and Osseous Site in Rat Materials and Methods

Nanohydroxyapatite and scaffold according to the invention were prepareas described in Example 1. The inventors assessed the implantation ofsaid scaffold in animal. Both the procedure and the animal treatmentcomplied with the Principles of Laboratory Animal Care formulated by theNational Society for Medical Research. The studies were carried out inaccredited animal facilities at the University of Bordeaux Segalen,under authorization (No.: 3300048 of the Ministere de l'Agriculture,France) and were approved by the Animal Research Committee of BordeauxUniversity.

Non-Osseous Implantation in Mice: Ectopic Bone Formation Analysis

The two different formulations of scaffolds: disk-shaped matriceswithout n-HA (Group 1) and the composite scaffold containing n-HA(MATRI+) (Group2) (cylinders of 4 mm diameter and 6 mm depth) wereinserted into subcutaneous pockets created in the dorsum of the12-week-old Balb/c mice weighing 25-30 g (Charles River Laboratories,France). Samples were retrieved after 15, 30 and 60 days of implantationand treated for micro-CT and histological analysis. Eight samples wereused for histological observation and micro-CT in each group.

Osseous Implantation in Rats: Orthotopic New Bone Formation Analysis

Medial holes, 5 mm diameter and 6 mm depth were created in both left andright femoral condyles of Wistar rats weighing 150-200 g (Charles RiverLaboratories, France) using trephine dental burr. Bone pieces wereremoved from the bone defect, the hole was rinsed with physiologicalsolution (NaCl 0.9% (w/v) before introducing the scaffold within thedefect. The two different scaffold formulations (matrices without n-HAand composite scaffold containing n-HA) were implanted into each bonedefect. A control experiment without scaffold was also conducted.Implants were retrieved 15, 30, 60 and 90 days after surgery and treatedfor micro-CT and histological analysis. Six samples were used formicro-CT and histological observation in each group.

Histological Procedure

At the end of each implantation period, animals were euthanized byinjecting an overdose of pentobarbital sodium (Nembutal®). Immediatelyafterwards, the implants and surrounding tissue were retrieved, fixedwith 4% (w/v) paraformaldehyde in a 0.1 M phosphate buffer and scannedwith micro-CT before histology. The samples were then prepared forhistological analysis. One part was decalcified, dehydrated and embeddedin paraffin. Thin sections (7 μm in thickness) were prepared and stainedwith hematoxylin and eosin and with Goldner's Trichrome for osteoidstaining. The other part were dehydrated in a graded series of ethanol,and then embedded with methylmethacrylate, which was subsequentlypolymerized. Ten to 15 μm transverse sections were made using a modifieddiamond blade microtome (Leica Microsystems SP1600, Rijswijk, TheNetherlands), with four sections obtained from each implant. Sectionswere stained with Goldner's trichrome, Von Kossa, and observed using aNikon Eclipse 80i microscope. Pictures were generated using a DXM 1200 C(Nikon) CCD camera.

Micro-Computed Tomography (Micro-CT)

Micro-CT was used to develop three-dimensional images of the implantsand surrounding tissue; these models were used to quantify the boneformation at each implant site. An ex vivo General Electric (GE)micro-CT (Explore LP Locus, General Electric), with a source voltage of80 kV, a current of 60 μA, and 15 μm resolution, was used to acquireX-ray radiographs. In vivo micro-CT (General Electric) was performedwith a source voltage of 150 mV, a current of 450 μA, and 45 μmresolution. After scanning, cross-sectional slices were reconstructedand 3D analyses were performed using Microview software. Each scanresult was reconstructed using the same threshold values to distinguishbone and air. Bone Mineral Content (BMC) and Bone Mineral density (BMD)volume were measured for each group and statistically analyzed using theStudent's t-test.

Protein Extraction from Subcutaneous Implants and ELISA Analysis ofOsteogenic and Angiogenic Growth Factors Retained within the Implants.

Subcutaneous implants retrieved after 2, 15, 30 and 60 days ofimplantation were crushed on ice with an electric crusher in PBScontaining a cocktail of protease inhibitors (10 μg/ml Aprotinine(Sigma), 10 μg/ml Leupeptin (Sigma) and 1 mM(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Fluka).The lysates were then centrifuged at 16 000 rpm and 4° C. for 20 min.The supernatant was collected and then frozen at −80° C. for ELISAanalysis. Quantification of the protein was performed usingbicinchoninic acid (BCA) protein assay kit (Thermoscientific) describedby Smith P K et al. (1985). Absorbance was read at 550 nm. There wereeight matrices without n-HA (Group 1) and composite scaffold MATRI+containing n-HA samples (Group 2), respectively for each time ofimplantation. The amounts of VEGF₁₆₅ and BMP2 retained within the twodifferent formulations of implants were quantified with the mouse VEGFimmunoassay kit (MMV00, Quantikine®, R&D systems), and BMP-2 immunoassaykit (DBP200, Quantikine CD, R&D systems), respectively.

X-Ray Diffraction Analysis

Subcutaneous implants of matrices without n-HA and composite scaffoldMATRI+ containing n-HA were retrieved after 15, 30 and 60 days ofimplantation. In order to obtain a fine powder without any organictissues, they were treated with bleach for 2 hours at room temperatureand then centrifuged to keep only the pellet. Structural properties wereexplored by X-ray diffraction (XRD) using PANalytical X'pert MPDdiffractometer (Bragg Brentano t-t geometry) equipped with a secondarymonochromator and uses a copper radiation (mean λ=1,5418 A°), theworking tension and intensity were 40 kV and 40 mA, respectively.

Samples were placed on a single-crystalline wafer sample holder made ofsilicium. Diffractograms were all measured with the same parameters:angular range from 8 to 80° (2t), step: 0.02°, measure time: one hour;Following X-ray diffraction (XRD) analysis of the material, phaseidentification through JCPDS-ICDD data (Diffract-Plus Eva Software,Bruker©) was compatible with a carbonated hydroxyapatite[Ca10(PO4)3(CO3)0.01(OH)1.3], displaying hexagonal lattice parameters(a=9.3892 A°; c=6.9019 A°; α=β=90° and g=120°; space group:P63/m(176)).

Statistical Analysis

All data were expressed as means±standard deviation (SD) and wereanalyzed using standard analysis of Student's t-test. Differences wereconsidered significant when p≦0.05 (a) or p≦0.01 (b).

Results

Two different scaffolds, matrices without n-HA (Group 1) and thecomposite scaffold MATRI+ containing n-HA (Group 2), were implanted inBalb/c mice for 15, 30 and 60 days. Micro-CT, quantification ofmineralization (BMC and BMD analysis) and histological studies wereperformed for both groups. Implantation of matrices without n-HA did notform any mineralized tissue from day 15 to day 60, as showed by micro-CT(FIG. 5A) and BMC and BMD quantification (FIG. 5B). In contrast,implantation in subcutaneous site of matrices containing n-HA (withoutany cells and growth factors) lead to the formation of a densemineralized tissue (FIG. 5A) as quantified by BMC (Bone Mineral Content)and BMD (Bone Mineral Density) measured at each time (FIG. 1B). Themineralization process starts at day 15 from the periphery of thescaffold (FIG. 1A) and lead to a high and dense mineralized tissue after60 days of implantation.

From histological data, the porous n-HA matrices exhibited favorablemineralized tissue responses at D15 and D60, as demonstrated by vonKossa staining of undecalcified sections of MATRI+ (FIG. 6A), comparedto matrix without n-HA. Von kossa staining is high after 60 days ofimplantation of MATRI+, compared to the same scaffold at day 15. Then-HA matrices before implantation stained with von kossa revealed aslight staining, due to the presence of the nanohydroxyapatite withinthe scaffold (not shown). However, the staining is much lower than thatobserved after 30 and 60 days of implantation.

Moreover, Goldner staining performed 60 days after implantation ondecalcified sections of MATRI+ (FIG. 6B), revealed, a dense fibrouscollagen tissue, mainly around the implant. Some collagen tissuepenetrate within the scaffold, exhibiting some lining osteoblast-likecells indicated by white arrows, in contact with the scaffold andnumerous vessels marked by black arrows on the histological picture. Noinflammatory event was detectable with both scaffolds, whatever the timeof implantation.

The XRD patterns of powder of n-HA matrices before implantation (D0) orretrieved at day 15 (D15) revealed specific peaks of hydroxyapatite atD15 on the spectrum (FIG. 7A). Peaks of Halite (H), probably due to thetreatment of the samples with bleach, were observed in all spectra. TheXRD patterns obtained at day 30 and day 60 were similar than thoseobserved at D15 for both groups (data not shown).

The inventors also explored whether the n-HA matrices compared tomatrices without n-HA could interact with endogeneous osteogenic andangiogenic growth factors. They have tested two major growth factorsthat play a fundamental role in angiogenesis and osteogenesis, theisoform VEGF₁₆₅ and BMP2, an osteoinductive factor that could, byitself, induces mineralization and bone formation. Two days ofimplantation, corresponding to the inflammatory phase observed followingmaterial implantation, both samples retained the two growth factors butto a different extent. Strikingly, the amount of BMP2 retained on MATRI+is 1.41 pg/μg protein extracted from the samples, while the matrixwithout n-HA retained only 0.12 pg/μg protein. For VEGF₁₆₅, the amountretained in MATRI+ and matrix without n-HA are 0.089 pg/μg protein and0.055 pg/μg protein, respectively. With time of implantation, and duringthe formation of the dense mineralized tissue, the concentration of BMP2(FIG. 8A) and VEGF₁₆₅ (FIG. 8B) decreased in both groups, compared todata obtained after 2 days, but remains significantly higher in theMATRI+ group after 30 and 60 days of implantation, compared to matrixwithout n-HA.

The scaffolds, matrices without n-HA (Group 1) and the compositescaffold MATRI+ containing n-HA (Group 2), were implanted in a criticalsize bone defect of 5 mm diameter and 6 mm depth in the femoral condyleof rats, for 15, 30 and 90 days. Micro-CT, quantification ofmineralization (BMC and BMD analysis) and histological analysis wereperformed for both groups. As showed by micro-CT, matrices with n-HA(MATRI+) (FIG. 9A) formed within the bone defect, a highly densemineralized tissue, compared to matrix without n-HA. Mineralizationincreases with time of implantation as shown by quantification analysisof the BMD and BMC (FIG. 9B) from day 15 to day 90 of implantation. BMCand BMD in the control group (empty) remain lower than in the othergroups, whatever the time of implantation.

Histological data after 90 days of implantation confirmed, a highstaining by von Kossa of the matrices with n-HA (MATRI+) compared withthe matrix alone without n-HA or the empty group (FIG. 10A). Goldnerstaining evidenced a fibrous tissue in the empty bone defect, while boneformation was enhanced within the MATRI+ implant after 90 days ofimplantation and occurred in direct contact of the MATRI+ implant (FIG.10B).

1-24. (canceled)
 25. A porous polysaccharide scaffold method obtainedby: i) preparing an alkaline aqueous solution comprising at least onepolysaccharide, a cross-linking agent and a porogen agent, ii)transforming the solution into a hydrogel by placing said solution at atemperature from 4° C. to 80° C. for a sufficient time to allowcross-linking of said at least one polysaccharide, iii) submerging saidhydrogel into a solvent, and iv) washing the porous polysaccharidescaffold obtained at step iii), wherein the alkaline aqueous solution ofstep i) further comprises hydroxyapatite.
 26. The porous polysaccharidescaffold according to claim 25, wherein the porogen agent is selectedfrom the group consisting of sodium chloride, calcium chloride, ammoniumcarbonate, ammonium bicarbonate, calcium carbonate, sodium carbonate,sodium bicarbonate and mixtures thereof.
 27. The porous polysaccharidescaffold according to claim 25, wherein a weight ratio of the at leastone polysaccharide to the porogen agent is in a range from 1:50 to 50:1.28. The porous polysaccharide scaffold according to claim 25, whereinsaid at least one polysaccharide is selected from the group consistingof dextran, pullulan, agar, alginic acid, starch, hyaluronic acid,inulin, heparin, fucoidan, chitosan and mixtures thereof.
 29. The porouspolysaccharide scaffold according to claim 25, wherein said at least onepolysaccharide is a mixture of pullulan/dextran in a ratio in a rangefrom 95:5 to 5:95.
 30. The porous polysaccharide scaffold according toclaim 25, wherein said at least one polysaccharide is a mixture ofpullulan/dextran/fucoidan in a ratio in a range from 70:20:10 to50:20:30.
 31. The porous polysaccharide scaffold according to claim 25,wherein said cross-linking agent is selected from the group consistingof trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl₃),epichlorohydrin, formaldehydes, carbodiimides, glutaraldehydes, andmixtures thereof.
 32. The porous polysaccharide scaffold according toclaim 25, wherein said hydroxyapatite is nano-hydroxyapatite.
 33. Theporous polysaccharide scaffold according to claim 32 wherein saidnano-hydroxyapatite is obtained from a solution of phosphoric acid at aconcentration between 0.3 to 1M, with a solution of calcium hydroxide ata concentration between 0.5 to 1.5M, through chemical precipitation atroom temperature.
 34. The porous polysaccharide scaffold according toclaim 32 wherein a concentration of nano-hydroxyapatite in the alkalineaqueous solution is between 0.01 and 10% (w/v).
 35. The porouspolysaccharide scaffold according to claim 25, wherein said porouspolysaccharide scaffold contains pores from 1 μm to 500 μm in size andthe porosity is from 4% to 75%.
 36. The porous polysaccharide scaffoldaccording to claim 25 wherein said solvent is an aqueous solution. 37.The porous polysaccharide scaffold according to claim 25, wherein aweight ratio of the at least one polysaccharide to said porogen agent isin a range from 1:30 to 30:1 (w/w).
 38. The porous polysaccharidescaffold according to claim 37, wherein said weight ratio is 75:25(w/w).
 39. The porous polysaccharide scaffold according to claim 37,wherein said ratio is selected from the group consisting of 70:20:10(w/w), 50:30:20 (w/w), and 73:22:5 (w/w).
 40. The porous polysaccharidescaffold according to claim 33, wherein said phosphoric acidconcentration is 0.6M and said calcium hydroxide concentration is 1M.41. The porous polysaccharide scaffold of claim 40, wherein saidnano-hydroxide concentration is between 0.1 and 0.5% (w/v) or between0.1 and 0.3% (w/v).
 42. The porous polysaccharide scaffold of claim 35,wherein the pores are from 10 to 200 μm in size and the porosity is from4% to 50%.